1. Field of the Invention
This invention relates to an additive preparation, the method of making the additive preparation and the method of using the additive preparation. The additive preparation is most particularly for use in blood collection devices wherein the additive preparation comes in contact with a blood specimen that has been previously treated with heparin. The additive preparation desirably comprises a formulation comprising heparinase and a carbohydrate. The additive formulation is desirably spray dried onto the inner wall of a blood collection device. In particular, the additive formulation of the present invention exhibits stability even when subjected to gamma irradiation.
2. Description of Related Art
Heparin is an anticoagulant that is used in surgical procedures and dialysis therapy to prevent clotting in intravenous lines and in the treatment of thrombolytic disorders. It affects thrombin activity by catalyzing the action of antithrombin III (ATIII), thereby preventing the conversion of fibrinogen to fibrin which results in clotting inhibition.
Heparin is also applied to a variety of clinical situations in addition to its use as an anticoagulant. It is used in hemodialysis treatments to prevent the blood from clotting during dialysis and as an antithrombotic agent in the treatment of deep venous thrombosis and orthopedic surgery. As a prophylactic of thrombosis, it is used in conjunction with prolonged intermittent intravenous administration of drugs and fluids. Recently, heparin also has been used in fibrinolytic therapy where it is co-administered with promoters of fibrin degradation such as tissue plasminogen activator, streptokinase or urokinase. Therefore, varying quantities of heparin are found in the blood of numerous hospitalized patients.
Patients receiving heparin therapy or who have been exposed to heparin through intravenous lines are frequently tested by a variety of means for the assessment of their hematological status, to monitor heparin therapy itself or for biochemical assays. However, the presence of heparin in blood specimens collected from heparinized patients causes several problems because the heparin interferes with clotting thereby rendering the results ambiguous or unobtainable. For biochemical assays, such problems include the prolongation of clotting and insufficient removal of fibrin thereby resulting in continual and unpredictable clot formation in specimens without added anticoagulants.
Furthermore, specimens with identifiable extended clotting times require additional handling and longer preparation times to remove or manage the heparin interference. In addition, if clots are not identified prior to assaying the specimen on an automated analyzer, clots forming within the instrument may lead to incomplete test results and/or instrument clogging. Therefore, testing accuracy is minimized, unnecessary instrument downtime may need to take place to unclog the instrument, additional specimens may need to be obtained to repeat the test and technical operator time is increased.
It is therefore desirable to resolve the heparin interference problem with a method that could expeditiously and specifically remove heparin from blood samples immediately after blood collection. The additive needed to accomplish this must function over a broad range of conditions. Such conditions include, but are not limited to, heparin being neutralized, quickly, while the additive itself, should not impart any effects on blood components over a lengthy exposure period. Furthermore, the treated samples containing heparin should give a result identical to untreated samples that have not been exposed to heparin.
Therefore, with the increasing demand for reducing turn around time and for fibrin free serum specimens there is a need to remove residual heparin from specimens collected from heparinized patients.
The present invention is a gamma irradiation stable additive formulation comprising a degradative glucanase enzyme specific for heparin and a stabilizer. The formulation may be effectively used as an additive in a tube to neutralize residual heparin in specimens taken from heparinized patients and accelerate clotting. In addition, the formulation is irradiation stable. The additive formulation is useful in effectively minimizing interference from heparin in a blood sample when used in a blood collection tube without interfering with the clinical analysis.
The additive formulation desirably comprises a degradative glucanase enzyme specific for heparin such as heparinase.
Desirably, the stabilizer of the additive formulation is to provide heparinase stability during controlled drying and elevated temperature storage of the formulation so that the heparinase can be stable.
The additive formulation preferably comprises heparinase and a disaccharide. Most preferably, the additive formulation comprises heparinase and trehalose.
An effective additive formulation compromising heparinase and trehalose, may be sprayed onto the wall of a tube and be rendered irradiation stable in the tube by controlled drying and prior removal of oxygen in the tube by backflushing with an alternate gas.
The additive formulation may further comprise a buffer solution so that the formulation resists changes in pH.
Most preferably, the additive formulation comprises:
(a) from about 50 IU/mL to about 80 IU/mL of degradative glucanase enzyme specific for heparin;
(b) from about 8 to about 12 weight percent of a stabilizer; and
(c) about 15 mL of a 150 millimolar (mM) buffer.
The unit heparinase is an International Unit (IU) which is the amount of heparinase which causes 1 micromole of double bonds to form per minute based on a molar extinction coefficient of about 5.1 at about 232 nm for the degradation products, the unsaturated uronic acids.
Most preferably, the additive formulation is used in a collection device such as a blood collection tube wherein the formulation is spray-dried onto the interior of the tube.
Additive formulations of the present invention are useful in providing neutralization of heparin and clotting of blood specimens.
Another attribute of the additive formulation is that it is stable when heated, dried and irradiated.
Advantages of the additive is that it achieves heparin neutralization of a blood sample of a heparinized patient faster and more completely than other available methods. Therefore, the additive formulation is useful in substantially removing heparin from a blood sample when used in a blood collection tube without interfering with the clinical analysis. The combination of the heparinase and trehalose in proper proportion is useful in eliminating the interference in clotting due to the presence of heparin. Therefore the additive formulation will help to deactivate residual heparin in specimens collected from heparinized patients. This method would reduce the handling of specimens from heparinized patients whereby current handling requires manual removal of latent fibrin from slow clotting specimens. Therefore, the method obviates the requirement for identifying problematic specimens or patient populations.
An advantage of the additive formulation comprising heparinase and trehalose and oxygen removal is a unique heat and radiation stable formulation. The formulation provides substantial recovery of about 50% to about 60% post irradiation.
An important attribute of the formulation of the present invention is that it exhibits stability even when subjected to gamma irradiation.
A method of protecting heparinase against denaturation during drying and irradiation, comprises the steps of incorporating an effective amount of trehalose into the heparinase. Therefore, according to the present invention there is provided a method of drying heparinase at a temperature above ambient, by incorporating trehalose into the heparinase which is to be spray dried, followed by oxygen removal prior to irradiation by back flushing the tube with a gaseous mixture of CO2/H2; 80:20 ratio.